Protein-based therapeutic products have contributed immensely to healthcare and constitute a large and growing percentage of the total pharmaceutical drugs. The majority of these FDA approved products are manufactured using mammalian cell culture systems. Over the past 10-20 years substantial progress has been made to overcome some of the key barriers to large-scale mammalian cell culture. Despite these improvements, the development of new biopharmaceutical products remains an expensive and lengthy process, where 20-30% of the total cost is associated with process development and clinical manufacturing. Currently most process optimization strategies are performed using a trial and error approach where cells are treated as a `black box'and process outputs are improved over several months by laborious experimentation. These empirical optimization techniques are widely used because in most cases little is known about the underlying physiological interactions that impact growth and protein production in the host cell lines. A fundamental understanding of cell line physiology and metabolism, enabled by computational modeling and simulation technologies, can greatly improve and accelerate media and process development in mammalian cell line systems. In the Phase I of this SBIR research project, we utilized our computational modeling platform and expertise in metabolic modeling and mammalian cell culture to reduce byproduct formation in a GS-NS0 murine myeloma cell line in collaboration with SAFC (Sigma-Aldrich Fine Chemicals) Biosciences. To implement our model-driven media optimization approach, we reconstructed a metabolic model for NS0 cell containing 456 metabolites and 470 metabolic reactions. The model was used to develop nutritional modifications to the basal media to reduce byproduct formation and improve growth and productivity. Experimental evaluation of our model-based media formulations in NS0 cell culture showed significant improvements over traditional methods for media analysis and resulted in approximately 12% lower lactate and up to 67% higher final product titers. With the successful completion of our Phase I proof-of-concept study, we now put forth a Phase II proposal that aims at completing the development of a commercial platform for media and process optimization that significantly improves the existing timelines associated with therapeutic protein production in mammalian cell lines. We plan to achieve this goal through: (1) refinement and expansion of the metabolic model of NS0 cell line, (2) integration of a transient flux balance approach for quantitative implementation of media designs, and (3) validation of the final framework using three case studies for antibody production in NS0 cell line. We will measure the overall success in Phase II by our ability to reduce the timelines to develop an optimized media that results in lower byproduct formation and higher productivity in cell culture. Successful completion of the specific aims outlined in the proposed plan will benchmark the commercial value of a model-driven approach in recombinant protein production through rational selection of nutrient supplementation and process optimization strategies. PUBLIC HEALTH RELEVANCE: Protein-based therapeutic products have contributed immensely to healthcare and constitute a large and growing percentage of the total pharmaceutical market. The majority of these FDA approved products are manufactured using mammalian cell culture systems. The proposed work aims at developing computational strategies that significantly improves the timelines and cost associated with therapeutic protein production in mammalian cells. Reducing the cost of therapeutic protein development and manufacturing would ensure that the next generation of medicines can be created in amounts large enough to meet patients'needs and at a price low enough that patients can afford them.